Biosynthetic cerebrospinal fluid control and method of use

ABSTRACT

The present invention relates to a biosynthetic cerebrospinal fluid control and method of use. Additionally, this invention relates to the isolation and purification of stable liquid human prealbumin, a component in the biosynthetic cerebrospinal fluid control.

This is a division of application Ser. No. 07/665,874, filed on Mar. 7,1991 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a stable biosynthetic liquid cerebrospinalfluid control and method of use. Additionally, this invention relates tothe isolation and purification of stable liquid human prealbumin, acomponent in the biosynthetic cerebrospinal fluid control.

2. Description of Related Art

Cerebrospinal fluid is formed by an ultrafiltration of the plasma.Normal values for cerebrospinal fluid analytes are not the same asplasma values. This difference is a result of the filtration processbeing selective and the fact that the chemical composition is adjustedby the blood-brain barrier. Analysis of this chemical composition is animportant diagnostic procedure. Disease increases cerebrospinal fluidprotein concentrations. Elevated cerebrospinal fluid total protein is anindicator of central nervous system pathology such as damage to theblood-brain barrier caused by meningitis or hemorrhage. IgG is theprimary immunoglobulin of cerebrospinal fluid. It is increased inseveral neurological conditions such as multiple sclerosis and viralmeningoencephalitis. Analysis of cerebrospinal fluid by serum proteinelectrophoresis is an important diagnostic test in the diagnosis ofmultiple sclerosis. Low glucose values signal infections such asbacterial, tuberculous or fungal meningitis. Low values are also seen asa result of infiltration of the meninges with malignant cells. Highlactic acid levels in cerebrospinal fluid indicate bacterial ortuberculous infection and rule out viral meningitis. Low cerebrospinalfluid chloride levels can be used as an indicator of tuberculousmeningitis.

Since the chemical composition of cerebrospinal fluid is similar toplasma comparable tests are performed. However, the levels of theseconstituents are not the same resulting in different normal values thanthose used for plasma. In order to assess the accuracy and precision ofthese diagnostic tests, a control similar to cerebrospinal fluid must berun. In the case of serum protein electrophoresis, a known proteincontrol is always run in a separate well. The protein fractions incerebrospinal fluid are not always clearly detected. Therefore, acontrol in which all the serum protein fractions are clearly defined isimportant. Most cerebrospinal fluid controls are prepared from actualspinal fluid. There are no tests, however, to detect the presence ofinfectious diseases in spinal fluid. Additionally, the recovery ofspinal fluid is difficult and expensive and the quality is varied. Othercerebrospinal fluid controls have been made from normal human bloodserum diluted with a diluent containing glucose and chloride ions, andthen lyopholized. Reconstitution of the control is then required beforeit can be used. See U.S. Pat. No. 3,753,925.

SUMMARY OF THE INVENTION

The present invention relates to biosynthetic cerebrospinal liquidcontrols based on human serum spiked with prealbumin. Two controls aredisclosed: one simulating normal spinal fluid and the second simulatingabnormal spinal fluid. The product is prepared from human serum andpurified human prealbumin in a buffer matrix formulated to simulatehuman cerebrospinal fluid. In particular, this invention relates to astable liquid human based cerebrospinal fluid control made by theprocess comprising: (a) combining a sufficient amount of lactic acid,chloride, glucose, serum, purified prealbumin, and potassium in a bufferto simulate normal human cerebrospinal fluid; (b) gassing said filteredfluid with oxygen to obtain normal electrophoretic pattern for humancerebrospinal fluid, and (c) filtering said fluid to remove allmicrobial contaminants.

The present invention also relates to high purity prealbumin and aprocess to make prealbumin. In particular, this invention relates to apurified prealbumin made by the process comprising: (a) diluting humanserum with a first buffer; (b) extracting globulins, ceroplasm andalbumin from normal serum diluted in a first buffer using ion exchangechromatography; (c) isolating the prealbumin containing fractions eludedfrom Step (b) by immunodiffusion; (d) pooling, concentrating and bufferexchanging the prealbumin containing fractions of Step (c) with a secondbuffer; (e) removing albumin from the said prealbumin containing pooledfractions of Step (d) by affinity chromatography; (f) isolating theprealbumin containing fractions eluted from Step (e) by immunodiffusion;(g) pooling and concentrating and buffer exchanging said prealbumincontaining fractions of Step (f) with a third buffer to increase theprealbumin concentration; (h) removing globulins from said pooledfractions of Step (g) by ion exchange chromatography; (i) pooling,concentrating and buffer exchanging with a fourth buffer to increaseprealbumin concentrations; (j) purifying the prealbumin containingfractions of Step (i) by gel filtration to remove any residual proteins;(k) isolating purified prealbumin fractions from Step (j) byelectrophoresis and immunodiffusion; and (l) pooling, concentrating andsterile filtering said purified prealbumin fractions of Step (k).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows protein electrophoresis of cerebrospinal fluid preparedaccording to this method.

FIG. 2 shows protein electrophoresis of cerebrospinal fluid preparedaccording to this method.

FIG. 3 shows protein electrophoresis of prealbumin prepared by thepresent method. Analysis by serum protein electrophoresis.

FIG. 4 shows an overlay of protein electrophoresis of prealbuminprepared by the present method, on to normal human serum pattern.

FIG. 5 shows protein electrophoresis of prealbumin prepared by the Razmethod; analysis by serum protein electrophoresis.

FIG. 6 shows an overlay of protein electrophoresis of prealbuminprepared by the Raz method, onto normal human serum pattern.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed invention involves diluting human serum with constituentsadjusted within ranges for cerebrospinal fluid. Cerebrospinal fluidcontains a very small amount of protein as compared to serum. Theprotein fractions are similar to those found in serum; however, forserum the quantity of prealbumin present is less than 1% whereas thequantity present in cerebrospinal fluid is 2 to 7% of the totalproteins. In order to increase the level of this protein, a prealbuminspike was added. This protein was effectively isolated from human serumusing column chromatography.

The product is formulated by the addition of the required constituentsto a 50 to 80 mM HEPES buffer matrix. The pH of the buffered matrix is7.3. Serum and prealbumin are added to the specifications required foreach level. Glucose, lactic acid, chloride, sodium, potassium are addedto obtain the desired concentrations as specified in Table I. Thebuffered solution is then gassed with 100% oxygen to remove apre-albumin fraction that migrates faster than prealbumin and thensterile filtered.

The assayed constituents for this product are: protein, glucose, lacticacid, chloride, sodium, potassium, immunoglobulins and protein fractionsby electrophoresis.

The Level I represents normal spinal fluid. Level II represents abnormalspinal fluid. The conditions observed in both levels of the control aremost commonly seen in meningitis, multiple sclerosis, and brain traumaor injuries.

                  TABLE I                                                         ______________________________________                                        Constituent Targets:                                                                                         LEVEL                                          PARAMETER  NORMAL    LEVEL I   II     UNITS                                   ______________________________________                                        Sodium     139-150   140-160   120-140                                                                              mmol/L                                  Potassium  2.7-3.9   2-4       3-6    mmol/L                                  Chloride   116-127   110-130    90-110                                                                              mmol/L                                  Lactic Acid                                                                              1.1-2.8   1-3       7-9    mmol/L                                  Glucose    45-80     45-80     25-40  mg/dL                                   Protein    15-45     15-45     50-80  mg/dL                                   ELECTROPHORETIC SEPARATION (% of Total Protein)                               PREALBUMIN 2-7       2-7       2-7    %                                       ALBUMIN    56-76     45-76     45-76  %                                       GLOBULINS:                                                                    ALPHA 1    2-7       2-7       2-7    %                                       BETA        7-18      7-18      7-18  %                                       GAMMA       7-14      7-19      7-19  %                                       IMMUNOGLOBULINS (RID)                                                         IgA          0-0.2   trace     trace  mg/dL                                   IgG        10-40      0-15      5-40  mg/dL                                   IgM          0-0.6   trace     trace  mg/dL                                   ______________________________________                                        Microbiology Specs: No growth to USP procedures.                          

EXAMPLE 1 Prealbumin Isolation from Serum

Units of normal human serum were pooled and the volume measured to beapproximately two liters. The pooled serum was diluted 50% in 50 mMpotassium phosphate buffer, pH 7.5, 0.1% azide. The diluted serum wassterile filtered through a 0.22 micron filter into sterile containers.The prepared serum was then loaded on to an ion exchange columncontaining DEAE Sephacel™ or DEAE Sepharose™ (Pharmacia) that has beenpreviously equilibrated with 50 mM potassium phosphate buffer, pH 7.5,0.1% azide. After completion of the sample load, the column was washedwith 50 mM potassium phosphate buffer, pH 7.5, 0.1% azide until an OD at280 nm is less than 0.2 as measured on an UV spectrophotometer. Thebound proteins were eluted with a gradient from 0 to 1M NaCl in 0.5Mpotassium phosphate buffer, pH 7.5, 0.1% azide. 12 mL fractions werecollected until the gradient was exhausted. This column removedceruloplasmin, globulins and albumin from the sample.

The fractions are tested by immunodiffusion for the presence ofprealbumin. When the fractions that contain prealbumin are identified,they are pooled, concentrated, and buffer exchanged with 20 mM potassiumphosphate buffer, pH 7.1, 0.02% azide. The pooled fractions wereconcentrated to a total protein of approximately 4 to 5 g/dL. Thefraction pool is then loaded on an affinity column containing Affi GelBlue™ (BIORAD) or Blue Sepharose™ (Pharmacia) which has beenequilibrated with 20 mM phosphate buffer, pH 7.1, 0.02% azide. Thischromatography media contains Cibacron Blue Dye F3G-A which has anaffinity for albumin. After the sample was loaded, the fractioncollector was started and 6 mL fractions were collected as the columnwas washed with 20 mM phosphate buffer, pH 7.1, 0.02% azide. As thesample was loaded, albumin binds to the blue dye and the remainingproteins passed through the column. The prealbumin containing fractionswere pooled, concentrated and buffer exchanged with 50 mM potassiumphosphate buffer, pH 7.5, 0.1% azide. The fractions were concentrated toa total protein of approximately 3 to 4 g/dL. The concentrated fractionswere then loaded onto an ion exchange column containing DEAE Sephacel™or DEAE Sepharose™ (Pharmacia) which has been equilibrated with 50 mMpotassium phosphate buffer, pH 7.5, 0.1% azide. The proteins were elutedusing a salt gradient of 0 to 1M NaCl in 50 mM potassium phosphatebuffer, pH 7.5, 0.1% azide. Fractions of 3 mL were collected until thegradient was exhausted. Fractions were tested for the presence ofprealbumin using immunodiffusion.

When the prealbumin containing fractions have been identified thesefractions were pooled, concentrated and buffer exchanged in 50 mMphosphate buffer with 170 mM sodium chloride, pH 7.5, 0.02% azide. Thefraction pool was concentrated to a total protein of approximately 2-7g/dL. This fraction pool was then loaded on a gel filtration columnwhich contained ULTROGEL® AcA 54 (IBF Biotechnics) equilibrated with 50mM phosphate buffer with 170 mM sodium chloride, pH 7.5, 0.02% azide.This column was then washed with 50 mM phosphate buffer with 170 mMsodium chloride, pH 7.5, 0.02% azide. Fractions were collected of 3 mLeach. Two protein peaks were collected. The prealbumin was mostlycontained in the second peak. Fractions were tested for the presence ofprealbumin using immunodiffusion. The fractions that contain prealbuminwere then tested by serum protein electrophoresis for the presence ofother serum proteins. The purified prealbumin fractions were selected,pooled and concentrated to a total protein of approximately 1 to 4 g/dL.These pooled fractions were sterile filtered and stored at 2°-8° C.

The purified prealbumin was tested for total prealbumin content usingserum protein electrophoresis, and radial immunodiffusion analysis forquantitative measurement of prealbumin. A single peak was observed andthe prealbumin was found to be 90 to 100% pure by proteinelectrophoresis. See FIG. 3. When compared to the electrophoreticpattern of normal serum, the peak is observed in the prealbumin regionand no other serum proteins are present. See FIG. 4. When spiked intonormal serum, the resulting electrophoretic pattern showed a peak in theprealbumin region. See FIG. 1 and 2. SDS PAGE electrophoresis shows asingle protein to be present. This protein is found in the correctmolecular weight range for prealbumin (54,000) The quantity ofprealbumin demonstrated yields of 80 to 100% depending on the purity ofprealbumin required. A commercially available prealbumin prepared fromhuman plasma using the method defined by Raz, A., et al., J. Biol.Chem., 244,12 (1969) was evaluated for purity. This prealbumin was foundto be only 75% pure by protein electrophoresis. See FIG. 5. Whencompared to the electrophoretic pattern of normal serum thecontaminating proteins are observed in the albumin, and alpha globulinregions. See FIG. 6.

The purified prealbumin has been monitored for stability while beingstored refrigerated and frozen. The prealbumin has been tested forquantity by radial immunodiffusion and purity by proteinelectrophoresis. After ten months storage at these conditions, theprealbumin has remained stable.

                  TABLE II                                                        ______________________________________                                        STABILITY OF PREALBUMIN                                                       MONTHS                                                                        ______________________________________                                                     STORAGE AT 2-8° C.                                        0            7395 mg of prealbumin/liter of solution                          4            7020 mg of prealbumin/liter of solution                          6            7879 mg of prealbumin/liter of solution                          10           7005 mg of prealbumin/liter of solution                          ______________________________________                                                     STORAGE AT -20° C.                                        0            N/A                                                              4            N/A                                                              6            7724 mg of prealbumin/liter of solution                          10           7275 mg of prealbumin/liter of solution                          ______________________________________                                    

EXAMPLE 2 Preparation of Cerebrospinal Fluid Control

A clean container with a stirring device is prepared. 800 mL ofdistilled is placed into the container. While mixing, the followingchemicals are added:

    ______________________________________                                        Constituents       Level I        Level II                                    ______________________________________                                        HEPES (N-2-hydroxyethyl                                                                          12.3   gm      9.2  gm                                     piperazine-N.sub.2 '-2-                                                       ethane sulfonic                                                               acid)                                                                         Sodium HEPES       9.4    gm      7.0  gm                                     Sodium Cloride     6.6    gm      5.3  gm                                     Potassium Cloride  0.19   gm      0.3  gm                                     Glucose            0.57   gm      0.33 gm                                     Sodium Lactate     0.38   gm      1.5  gm                                     Human Serum        0.29   gm      0.63 gm                                     Prealbumin         10.0   mg      15.0 mg                                     Sodium Azide 25%   0.8    mL      0.8  mL                                     ______________________________________                                    

After all chemicals are dissolved, the total volume of the solution isbrought to one liter with distilled water. All constituents are analyzedand adjusted within the above described specifications. A gas cylinderof oxygen is connected to a two stage regulator. Rubber tubing orequivalent is connected to the regulator and to the batching container.The first stage of the regulator is opened. The second stage is slowlyopened until the gas flow through the solution is approximately 0.4 SCFH(square cubic feet per hour). While mixing, the pool is flushed in thismanner at room temperature.

After flushing, a sample of the solution is removed and concentratedapproximately 60 times. This concentrated sample is then evaluated byserum protein electrophoresis. If the electrophoretic pattern does notshow a single peak in the prealbumin region, reflushing is necessary.

After a normal electrophoretic pattern is recovered, the solution issterile filtered through 0.22 micron membranes into sterilizedcontainers. The sterile solution is then filled into sterilized vials atthree mL each.

These cerebrospinal fluid controls were evaluated for stabilityaccording to a protocol for the evaluation of the stability ofdiagnostics products. This protocol states guidelines for acceleratedstability studies. According to this protocol, a product that is storedat 37° C. for one week is stable for one year at 2°-8° C. Acceleratedstability studies were used to determine the performance characteristicsof the product under storage conditions which stress the product incomparison to those recommended for use and handling of the product. Thecerebrospinal fluid controls were analyzed after storage at 25° C. forthree months and 37° C. for four weeks. Results from these analyses showthe product to be stable and therefore have a predicted shelf life ofgreater than three years. The product has been monitored at 2°-8° C. forgreater than one year. See Table III.

                  TABLE III                                                       ______________________________________                                        STABILITY OF CEREBROSPINAL FLUID CONTROL                                      ______________________________________                                        CON-                  2-8° C.                                                                         25° C.                                                                        37° C.                           STITUENTS UNITS       Storage  Storage                                                                              Storage                                 ______________________________________                                        LEVEL I                                                                       PROTEIN   mg/dL       28       30     28                                      LACTIC    mM          1.2      1.1    1.2                                     ACID                                                                          GLUCOSE   mg/dL       56       56     56                                      CHLORIDE  mM          120      127    122                                     SODIUM    mM          149      150    149                                     POTASSIUM mM          2.6      2.6    2.6                                     IgA       mg/dL       1.2      1.1    1.3                                     IgG       mg/dL       4.6      5.0    4.3                                     IgM       mg/dL       1.2      1.6    1.4                                     ELECTRO-                                                                      PHORESIS:                                                                     PRE-      % OF TOTAL  6.2      6.2    5.2                                     ALBUMIN                                                                       ALBUMIN   % OF TOTAL  66       65     66                                      ALPHA 1   % OF TOTAL  3.2      3.0    3.7                                     ALPHA 2   % OF TOTAL  6.3      6.3    6.2                                     BETA      % OF TOTAL  7.7      8.2    8.2                                     GAMMA     % OF TOTAL  10.5     11.2   11.6                                    LEVEL II                                                                      PROTEIN   mg/dL       61       66     64                                      LACTIC    mM          7.6      7.7    7.6                                     ACID                                                                          GLUCOSE   mg/dL       31       34     33                                      CHLORIDE  mM          102      106    102                                     SODIUM    mM          127      127    127                                     POTASSIUM mM          4.1      4.2    4.1                                     IgA       mg/dL       2.5      2.5    3.0                                     IgG       mg/dL       10.2     10.4   10.3                                    IgM       mg/dL       1.9      2.4    1.9                                     ELECTRO-                                                                      PHORESIS:                                                                     PRE-      OF TOTAL    5.4      4.9    4.6                                     ALBUMIN                                                                       ALBUMIN   % OF TOTAL  63       63     62                                      ALPHA 1   % OF TOTAL  2.8      2.4    2.7                                     ALPHA 2   % OF TOTAL  7.4      7.6    7.8                                     BETA      % OF TOTAL  8.6      9.6    9.6                                     GAMMA     % OF TOTAL  12.5     12.6   13.5                                    ______________________________________                                    

The cerebrospinal fluid controls were also evaluated for open vialstability. Vials were tested after being open for two weeks. Analyses ofthe opened vials showed no change when compared to vials that werefreshly sampled. See TABLE IV.

                  TABLE IV                                                        ______________________________________                                        CON-                  FRESH                                                   STITUENT    UNITS     VIAL      OPEN 14 DAYS                                  ______________________________________                                        OPEN VIAL STABILITY LEVEL I                                                   Protein     mg/dL     25.5      25.7                                          Glucose     mg/dL     60.0      60.2                                          Sodium      mM        158       158                                           Chloride    mM        113       112                                           IgG         mg/dL     4.98      4.99                                          IgA         mg/dL     1.18      1.16                                          IgM         mg/dL     <0.69     <0.69                                         ELECTROPHORESIS:                                                              Prealbumin  % of Total                                                                              3.6       4.4                                           Albumin     % of Total                                                                              65        64                                            Alpha 1     % of Total                                                                              3.0       3.6                                           Alpha 2     % of Total                                                                              6.8       7.1                                           Beta        % of Total                                                                              9.2       9.2                                           Gamma       % of Total                                                                              11.9      12.1                                          OPEN VIAL STABILITY LEVEL II                                                  Protein     mg/dL     59.5      59.1                                          Glucose     mg/dL     33.3      33.2                                          Sodium      mM        127       127                                           Chloride    mM        96        97                                            IgG         mg/dL     11        11                                            IgA         mg/dL     2.59      2.58                                          IgM         mg/dL     0.89      0.91                                          ELECTROPHORESIS:                                                              Prealbumin  % of Total                                                                              2.5       2.6                                           Albumin     % of Total                                                                              61        62                                            Alpha 1     % of Total                                                                              4.0       4.1                                           Alpha 2     % of Total                                                                              8.5       7.8                                           Beta        % of Total                                                                              10.0      9.5                                           Gamma       % of Total                                                                              13.5      13.8                                          ______________________________________                                    

EXAMPLE 3

The cerebrospinal fluid control prepared in Example 2 was used as acontrol in several diagnostic tests. The results of these assays arereported in TABLE V.

                  TABLE V                                                         ______________________________________                                        METHODS COMPARISON                                                            CON-                                                                          STI-                           LEVEL  LEVEL                                   TUENT   UNITS    METHOD        I      II                                      ______________________________________                                        Protein mg/dL    DuPont aca 4  28.5   59.8                                            mg/dL    DuPont aca 3  22     57                                              mg/dL    Kodak Ektachem                                                                              25.6   75.6                                            mg/dL    Abbott Spectrum                                                                             28.6   60.2                                    Lactic Acid                                                                           mM       DuPont aca 3  1.2    7.0                                             mM       Baxter Paramax                                                                              1.4    7.1                                     Glucose mg/dL    DuPont aca 4  57.2   33.5                                            mg/dL    DuPont aca 3  57     33.8                                            mg/dL    DuPont Dimension                                                                            56.6   33.4                                            mg/dL    Kodak Ektachem                                                                              60.1   35.9                                            mg/dL    Abbott Spectrum                                                                             58.5   35.4                                            mg/dL    Baxter Paramax                                                                              60.0   36.0                                    Chloride                                                                              mM       DuPont aca 3  122    104                                             mM       Kodak Ektachem                                                                              112    93                                              mM       Abbott Spectrum                                                                             119    101                                             mM       DuPont Dimension                                                                            114    97                                              mM       NOVA Biomedical                                                                             119    101                                             mM       Baxter Paramax                                                                              113    95                                      Sodium  mM       Abbott Spectrum                                                                             152    130                                             mM       DuPont Dimension                                                                            153    128                                             mM       NOVA Biomedical                                                                             150    126                                     Potassium                                                                             mM       NOVA Biomedical                                                                             2.7    4.1                                     IgG     mg/dL    RID           8.0    16                                              mg/dL    Beckman Array 5.0    11                                      IgA     mg/dL    RID           1.4    3.0                                             mg/dL    Beckman Array 1.2    2.6                                     IgM     mg/dL    RID           1.4    1.9                                             mg/dL    Beckman Array <0.69  0.91                                    ELECTROPHORESIS: % OF TOTAL                                                   HELENA                                                                        Prealbumin             7.0      5.5                                           Albumin                63       61                                            Alpha 1                3.8      3.9                                           Alpha 2                6.4      7.4                                           Beta                   7.3      8.3                                           Gamma                  13.0     14.0                                          BECKMAN PARAGON                                                               Prealbumin             5.5      3.5                                           Albumin                66       67                                            Alpha 1                3.7      3.7                                           Alpha 2                6.6      7.1                                           Beta                   7.6      7.6                                           Gamma                  10.0     11.0                                          ______________________________________                                    

We claim:
 1. An electrophoretic method to assess human cerebrospinalfluid comprising:a) preparing a stable liquid human based cerebrospinalfluid control, wherein the control is made by the process comprising:(i)combining a sufficient amount of lactic acid, chloride, glucose, serum,purified prealbumin, and potassium in a buffer to simulate normal humancerebrospinal fluid; (ii) gassing said fluid with oxygen to obtainnormal electrophoretic patter for human cerebrospinal fluid; and (iii)filtering said fluids to remove all microbial contaminants; b. obtainingan electrophoretic pattern of said control and a sample humancerebrospinal fluid; and c. comparing the electrophoretic pattern of thecontrol with the electrophoretic pattern of the sample.
 2. The method ofclaim 1 wherein the electrophoretic patterns of the sample and controlare obtained simultaneously.